Liquid microcolumn chromatograph

ABSTRACT

The liquid microcolumn chromatograph comprises a microcolumn (1), unit (2) for feeding eluent, made as a reservoir (3) separated by a &#34;slack&#34; diaphragm (4) into two half-chambers (5,6) one whereof communicates with a source (7) of pressure and the other is connected to a source (8) of eluent and to an inlet into the microcolumn (1) a flow-through cell (9), and an eluent-collecting vessel (14). The chromatograph is provided with an additional reservoir (10) separated by a &#34;slack&#34; diaphragm (11) into two half-chambers (12, 13) and is also provided with an additional source (15) of pressure, the half-chamber (12) being communicated with an outlet of the flow-through cell (9) and connected to the eluent-collecting vessel (14), whereas the half-chamber (13) communicates with the additional source (15) of pressure.

FIELD OF THE ART

The present invention relates to analytical instrumentation and hasspecific reference to the design of liquid microcolumn chromatographs.

PRIOR ART

Known in the art is a liquid microcolumn chromatograph comprising amicrocolumn, a unit for feeding eluent in the form of a reservoirseparated by a "slack" diaphragm into two chambers of which onecommunicates with a source of pressure and is filled with a workingagent and the other chamber communicates with a source of eluent andwith an inlet into the microcolumn, a flow-through cell of a detectorlocated at the outlet of the microcolumn, and an eluent receiver (SU, A,715996).

In microcolumn chromatography columns with sorbent are used, theparticle size of the sorbent being on the order of units of microns. Thepressure gradient in such columns reaches hundreds of atmospheres. Ahigh pressure gradient in the microcolumn makes the etuent flow throughthe heterogenous packing of the column non-uniform. High pressure surgesset up when the components of the sample are passing through the sorbentof the microcolumn fail to coincide with the time of passage of thesecomponents through the detector cell. Consequently, the detector (whichis commonly a high-sensitive one of the refractometer type) recordsblurred boundaries of chromatographic peaks, corresponding to eachcomponent, i.e. the shape of the peaks fails to represent the actualcomposition of the sample. Moreover, bubbles of the solute gas liberatedfrom the eluent due to the sharp drop in pressure brought about by thedescent of the eluent down the chromotographic microcolumn on the onehand, block sorbent channels and, on the other hand, are recorded poresby the detector and interfere with the useful signal. As a result, theaccuracy of chromatographic analysis is impaired.

To eliminate the above-stated disadvantages in liquid chromatographs, itis known to employ a means of setting up a backpressure at the outlet ofthe chromatographic column. Thus, a liquid chromatograph is known,wherein the backpressure at the column outlet is provided in the form ofa liquid column (JP, B, 54-19194).

However, the known means of providing backpressure cannot be used inconjunction with liquid microcolumn chromatographs which require thatthe outlet pressure in the microcolumn should be of the same order asthe inlet one-commonly of a significant value as already mentionedabove. Only on condition that this requirement is met the pressurefluctuations in the microcolumn can amount to fractions of percent ofthe pressure in the cell of the detector. To make a liquid columncapable of ensuring a requisite outlet pressure in a microcolumn is notfeasible.

DISCLOSURE OF THE INVENTION

It is an object of the invention to provide a liquid microcolumnchromatograph which would be equipped with a means for setting up abackpressure at the outlet of the microcolumn thereof, featuring asimple and reliable design, which would enable the elimination ofevolution of gas bubbles at the microcolumn outlet, as well asfluctuations of pressure in the microcolumn and, in the final run,enable an increase in the confidence and accuracy of chromatographicanalysis.

The essence of the invention consists in that a liquid microcolumnchromatograph comprising a microcolumn, a unit for feeding eluentdesigned as a main reservoir separated by a "slack" diaphragm into twohalf-chambers of which one communicates with a main source of pressureand is filled with a working agent and the other half chambercommunicates with a source of eluent and with an inlet to themicrocolumn, a flow through cell of a detector at the microcolumnoutlet, and an eluent receiver, according to the invention, is providedwith an additional reservoir separated by a "slack" diaphragm into twohalf-chambers and given a volume which is essentially greater than thevolume of the eluent needed for one analysis and is also provided withan additional source of pressure, one half-chamber of the additionalreservoir communicating with an outlet of the flow-through cell of thedetector and with the eluent receiver and the other half-chamber of theadditional reservoir communicates with the additional source ofpressure.

It is expedient that the main and additional reservoirs should beidentical in design, the inside surfaces of their half-chambers beshaped each as a body of revolution, and the surface area of the "slack"diaphragm be equal to or greater than the area of the inside surface ofthe half chamber.

It is also expedient that a pickup of the position of the "slack"diaphragm be provided in each of the half-chambers communicating withthe corresponding source of pressure.

The pickup of the position of the "slack" diaphragm may be made in theform of a cylindrical hollow projection on the surface of thehalf-chamber, which is fitted with signalling elements indicating thetwo extreme positions of the "slack" diaphragm and contains a rodattached to the centre of the "slack" diaphragm with one rod endcarrying a signalling element indicating a current position of the"slack" diaphragm at the other end, the rod being installed with apossibility of moving inside the projection and interacting by thesignalling element thereof with the signalling elements of theprojection.

At the outlet of the flow-through cell of the detector in the liquidmicrocolumn chromatograph according to the invention there is located ameans of setting up a backpressure at the outlet of the flow-throughcell of the detector which is of a value commensurable with the value ofthe pressure at the inlet to the chromatographic column. Owing to thisbackpressure, the pressure surges resulting from the passage of samplecomponents through the sorbent of the microcolumn are neigligibly smalland no gas bubbles are formed at the outlet from the column. Apart fromthat, any pressure surges which are set up are snubbed owing to thepresence of the additional voluminous reservoir at the outlet from theflow-through cell. Since all the eluent needed for analyzing thousandsof samples is contained between the two "slack" diaphragms whichinteract with the working fluid found under the same pressure allpressure surges arising in the hydraulic system of thechromatograph--such as those due to the jerkwise feeding of the eluentby step motor-actuated syringe pumps, operation of a sample-feedingstopcock and other automatic stopcocks--are snubbed. As a result, anypossibility of generating spurious signals, distorting the shape ofchromographic peaks and that of detector background is eliminated, theaccuracy and sensitivity of the detector are increased.

BRIEF DESCRIPTION OF THE DRAWING

The sole figure shows a general schematic view of the liquid microcolumnchromatograph according to the invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Referring to the drawing, the liquid microcolumn chromatograph comprisesa microcolumn 1 packed with a fine sorbent, a unit 2 for feeding eluentwhich is provided in the form of a reservoir 3 separated by a "slack"diaphgragm 4 into two half-chambers 5 and 6, whereby the half-chamber 5communicates with a source 7 of pressure--e.g. a syringe pump--and isfilled with a working agent and the half chamber 6 communicates with asource 8 of eluent and with an inlet to the microcolumn 1. Connected toan outlet from the microcolumn 1 is a flow-through cell 9 of a highlysensitive laser detector (not shown), which is also connected to anadditional reservoir 10 the volume whereof is essentially greater thanthat of the eluent needed for one analysis. The additional reservoir 10is separated by a "slack" diaphragm 11 into two half-chambers 12 and 13,whereby the half-chamber 12 communicates with the flowthrough cell 9 andwith an eluent-collecting vessel (receiver) 14 and the half-chamber 13communicates with an additional source 15 of pressure (e.g. a syringepump or a cylinder with gas). The half-chamber 13 is filled with aworking agent (an inert incompressible lubricating liquid or a gas). Thesources 7, 15 of pressure, provided they are syringe pumps (as shown inthe drawing), are connected each to an auxiliary reservoir 16 containingthe working agent which is used to refill the delivery ends of the pumpsafter delivery strokes for injecting pressurized liquid. Precisionsyringe pumps feature ultrasmall deliveries (units of microlitres perminute per stroke) and cannot have voluminous delivery ends. Thereforetheir their regular refilling is indispensable.

The working agent-filled half-chambers 5, 13 are provided each with adrain vessel 17 usable during the adjustments of the diaphragms 4, 11for position in the reservoirs 3, 10. The half-chambers 5, 13 with theworking agent and the half-chambers 6, 12 with the eluent are shaped asbodies of revolution, and the surface area of the "slack" diaphgragms 4,11 equals or is greater than the area of the inside surface of each ofthe half-chambers 5, 6, 12 and 13.

The half-chambers 5, 13 with the working agent are provided each with ahollow cylindrical projection 18 on their outside surface, theprojection 18 being provided with a pressure transducer 19. Eachprojection 18 is provided with two signalling elements 20 serving tomonitor the two extreme positions of the "slack" diaphragms 4, 11 insidethe reservoirs 3, 10. Two rods 21 capable of moving freely in thecylindrical projections 18 are linked each to the centres of thecorresponding "slack" diaphragms 4 and 11. At the opposite end of eachrod 21 a signalling element 22 is provided which generates a signal inthe element 20 at the instant the element 22 is located opposite theelement 20. A remotely-operated stopcock 23 is provided at the deliveryend of each of the sources 7, 15 of pressure. Similar remotely-operatedstopcocks 24 are provided at the eluent-containing reservoirs 8, 14, andremotely-operated stopcocks 25, 26 are provided at the inlet to and atthe outlet from the chromatographic column 1, respectively. The stopcock25 is located upstream of a metering means 27 which is secured directlyto the inlet end face of the microcolumn 1, and the stopcock 26 islocated downstream of the flow-through cell 9 which is also secureddirectly to the outlet end face of the microcolumn 1.

Hand-operated stopcocks 28 which can be set either "open" or "closed"are provided on the lines connecting the chambers 5, 13 with the drainvessels 17.

The pressure trensducers 19, the signalling elements 20, an electricdrive 29 of the sources 7, 15 of pressure and the actuators of theremotely-operated stopcocks 23, 24, 25, 26 are connected to a controlcomputer 20.

The liquid microcolumn chromatograph operates as follows. Thehalf-chamber 6 of the unit 2 for feeding eluent is refilled with a fresheluent and the half-chamber 12 of the additional reservoir 10 is drainedof the eluent spent during the preceding analyses, obeying the signalsof the computer 30.

The motive force for the operations of redistributing liquids in thechromatograph is provided by the sources 7, 15 of pressure exertingtheir action on the inert working agent in the half-chambers 5 and 13. Adischarge of the working agent from the half-chambers 5, 13 initiatesthe refilling of the chambers 6, 14 with the eluent from the vessels 8,14, provided the stopcocks 24 have been set open by a signal from thecomputer 30. The eluent contained in the half-chambers 5, 12 is admittedinto the chromatographic column 1 owing to the delivery of the workingagent either into the half-chamber 5 or the half-chamber 13, dependingon the direction of admitting the eluent. The pressure sustained by theworking agent is applied to the eluent by way of either the diaphragm 4or the diaphragm 11, and the rate of feeding the working agent equalsthe rate of pumping the eluent through the packing of the microcolumn 1.Drained of the eluent is that of the half-chambers 6, 12 which isexposed to a higher pressure, the other half-chamber being filled at thesame time. To that end, the syringe pumps (the sources 7, 15 ofpressure) must operate in antiphase. The pump ensuring a higher pressuremust be on a delivery stroke, while the pump at the opposite end of themicrocolumn 1 must be on a suction stroke (the plunger would movedownwards).

Since the delivery ends of the syringe pumps have a small volume, anumber of refilling-draining cycles of the delivery end of the source 7of pressure are required before the half-chamber 6 is filled with theeluent in an amount sufficient for carrying out several thousands ofanalyses of the chromatograph.

Concurrently with the above operations, the delivery end of the source15 of pressure is refilled with the working agent in order to have thisagent in stock in an amount required for expelling the spent eluent fromthe half-chamber 12 into the eluent-collecting vessel 14.

The procedure of stocking the chromatograph with fresh eluent is asfollows. The stopcocks 25, 26, 28 are closed and the stopcocks 24 areopened. The source 7 of pressure (syringe pump) begins a suction stroke(the pluger moves downwards) so that the working agent contained in thehalf-chamber 5 enters the delivery end of the pump. The line connectingthe delivery end of the pump to the auxiliary reservoir 16 is closed bythe stopcock 23. As soon as the delivery end of the pump is filled withthe working agent, a signal from the computer 30 causes the stopcock 23to open the line connecting the pump to the auxiliary reservoir 16 andto close the line from the pump to the half-chamber 5. Another signal ofthe computer 30 sets the electric drive 29 of the pump into operationwhich ejects the liquid from the delivery end into the auxiliaryreservoir 16. When the stopcock 23 connects the delivery end of the pumpto the half-chamber 5, the pump draws another portion of the workingagent from the half-chamber 5. During these cycles similar amounts ofthe fresh eluent contained in the source 8 of eluent are drawn into thehalf-chamber 6.

The position of the diaphragm 4 indicative of the amounts of the workingagent and eluent in the half-chambers 5 and 6, respectively, ismonitored by the signalling elements 20 when they are passed by thesignalling element 22 attached to the rod 21. The rod 21 reciprocatesfreely within the cylindrical projection 18 being acted upon by thediaphragm 4 it is linked to. When the bulk of the working agent has beendrawn from the half-chamber 5 and the rod 21 has entered the interior ofthe projection 18 so that the signalling element 22 at the end of therod 21 registers with the signalling element 20 of the projection 18,the pump (i.e. the source 7 of pressure) is automatically stopped by thecomputer 30. The reservoir 3 is then filled with eluent in an amountsignificantly exceeding (thousand-fold) the quantity needed for a singleanalysis.

Likewise, the half-chamber 12 of the additional reservoir 10 is beingdrained, provided the stopcocks 25, 26, 28 are closed while the stopcock24 is open. The eluent accumulated in the half-chamber 12 is expelled bythe diaphragm 11 into the eluent-collecting vessel 14, whereas thesource 15 of pressure delivers the working agent into the half-chamber13. The delivery end of the syringe pump, if one is being used, isfilled with the working agent from the auxiliary reservoir 16 during theback stroke of the pump, provided the stopcock 23 is set so as toconnect the delivery end of the pump to the auxiliary reservoir 16 andthe line connecting the pump end to the half-chamber 13 is closed. Thesignal indicative of the extreme positions of the diaphgragm 11 comesfrom the signalling elements 20 in the same way as this was describedreferring to the diaphragm 4. At the end of draining the reservoir 10,this becomes filled with the working agent and the spent eluent isaccumulated in the eluent-collecting vessel 14. In response to signalsof the computer 30, the stopcocks 25, 26 protecting the microcolumn 1are opened, the stopcocks 24 are closed and the stopcocks 23 are set soas connect the filled delivery ends of the pumps (the sources 7, 15 ofpressure) to the half-chambers 5 and 13. The source 7 of pressureinduces a flow of eluent through the column 1 and the source 15 ofpressure sets up a backpressure at the outlet from the column 1 which isof a value providing for a given outlet-to-inlet pressure ratio. Thepressure transducers 19 provided in the cylindrical projections 18measure the inlet and outlet pressures in the column 1. As soon as thegiven values of these pressures (say 200 or 100 atm) are attained, asample is introduced into the column 1 with the aid of the meteringmeans 27. The separation process takes then place, and the separatedsolutes are detected and measured while passing through the chromotank9. The microcolumn 1 is rinsed by circulating fresh eluent therethrough,and the next sample is injected. The draining vessels 17 with thestopcocks 28 are used for adjusting the chromatograph, e.g. forequalizing the positions of the diaphragms 4, 11 in the reservoirs 3, 10or for draining some working agent when the source 15 of pressure is acylinder with gas.

If the sources 7, 15 of pressure are identical syringe pumps, these mustoperate in antiphase. This means that at the time one of the pumps is ona delivery stroke, injecting eluent into the column 1, the other pumpmust be on a suction stroke at the same speed (provided the givenbackpressure has been set up during the preparation of the system).

More often than not working agent is delivered in small amounts and isregularly drained into the draining (vessel 17). The detector backgroundis noted every time.

A salient feature of the herein-proposed liquid microcolumnchromatograph is that the microcolumn 1 is connected at both ends to thereservoirs 3, 10 the volume whereof is tenths thousands times greaterthan the free volume of the microcolumn 1. Moreover, the reservoirs 3,10 are always filled with liquids sustaining high pressure. Comparedwith this pressure, the pressure surges resulting from the operation ofthe step motors of the electric pump drives 29 or from the elution of asample through the microcolumn 1 amount to small fractions of percent.The large volumes of liquids snub the pressure surges. A delivery of aportion of liquid needed for a signal analysis from the unit 2 forfeeding the eluent into the additional reservoir 10 changes the pressurein this last-named reservoir only by about 0.01%, i.e. by an amountwhich is outside the range of detector sensitivity.

Also on the positive side of the chromatograph according to theinvention is the equalization of the temperature of the eluent beforeand after the microcolumn 1. In other words, no difference existsbetween the temperature of the cell 9 and that of the eluent which is afactor of importance if use is made of refractometers. Large volumes ofliquids before and after the microcolumn 1 cannot have an appreciabletemperature gradient. Therefore an adequate thermal stability isprovided to obtain accurate measurements. To that end it is goodpractice to make both reservoirs 3, 10 of a material displaying goodthermal conductivity and link them by structural elements of the samekind which would also surround the microcolumn 1 and the flow-throughcell 9. Most of the chromatographs meet this requirement and theirmicrocolumn 1 is either in metal or is provided with a metal casing.

The present liquid microcolumn chromatograph finds in the analysis ofcomplex mixtures of organic compounds when the sample for the test inquestion is too small for a conventional treatment. This may be thecase, for example, in medicine in connection with analyzing smears takenfrom organs of recently born babies. Likewise, in genetic engineering aneed may arise to determine the structure of hormones and enzymes, bothsynthetic and produced in the body. Determining the amount of pesticidesin insects is a problem confronted with in agriculture. Criminologyresorts to liquid microcolumn chromatography and so do various domainsof science and engineering.

What is claimed is:
 1. A liquid microcolumn chromatograph comprising amicrocolumn (1), a unit (2) for feeding eluent, said unit being made asa main reservoir (3) separated by a "slack" diaphragm (4) into twohalf-chambers (5, 6) of which one communicates with a main source (7) ofpressure and is filled with a working agent, while the other isconnected to a source (8) of eluent and to an inlet to the microcolumn(1), a flow-through cell (9) of a detector at the outlet of themicrocolumn (1), and an eluent-collecting vessel (14), c h a r a c t e ri z e d in that it is provided with an additional vessel (10) whosevolume is essentially greater than that of the eluent required forcarrying out a single analysis, separated by a "slack" diaphragm (11)into two half-chambers (12, 13), and with an additional source (15) ofpressure, the half-chamber (12) communicating with the outlet of theflow-through cell (9) of the detector and with the eluent-collectingvessel (14), whereas the half-chamber (13) is communicated with anadditional source (15) of pressure.
 2. A liquid microcolumnchromatograph according to claim 1, c h a r a c t e r i z e d in thatthe main and additional reservoirs (3, 10) are identical and the innersurface of the half-chambers (5, 6, 12, 13) thereof is a body ofrevolution, whereas the surface area of the "slack" diaphragm (4, 11) isequal to or greater than the area of the inner surface of thehalf-chamber (5, 6, 12, 13).
 3. A microcolumn chromatograph according toclaim 1 or 2, c h a r a c t e r i z e d in that in each half-chamber (5,13), communicated with its source (7, 15) of pressure, a pickup of theposition of the "slack" diaphragm (4, 11) is arranged.
 4. A microcolumnchromatograph according to claim 3, c h a r a c t e r i z e d in thatthe pickup of the position of the "slack" diaphragm (4, 11) is made as ahollow cylindrical projection on the surface of the half-chamber (5, 13)and is equipped with signalling elements (20) monitoring two extremepositions of the "slack" diaphragm (4, 11), and a rod (21) whose one endis linked with the centre of the "slack" membrane (4, 11) and whoseother end carries a signalling element (22) indicating a currentposition of the "slack" diaphragm (4, 11), said rod being movable withinthe projection (18) and interacting by its signalling element (22) withthe signalling elements (20) of the projection 18.